Asian-Aust. J. Anim. Sci.
Vol. 20, No. 6 : 894 - 899 June 2007
www.ajas.info
Effects of Plantain (Plantago lanceolata L.) Herb and Heat Exposure on Plasma Glucose Metabolism in Sheep
M. Al-Mamun1,C. Tanaka1, Y. Hanai1, Y. Tamura2andH.Sano1,* 1Faculty of Agriculture, IwateUniversity,Ueda 3-18-8, Morioka 020-8550, Japan
* Corresponding Author: H. Sano. Tel: +81-19-621-6165, Fax: +81-19-621-6165, E-mail: sano@iwate-u.ac.jp
2 National Agricultural Research Center for the Tohoku Region, Morioka, Japan.
Received October 4, 2006; Accepted February 7, 2007
ABSTRACT :An experiment was conducted using a [6, 6-2H]glucose isotope dilution method to determine the effects of plantain (Plantago lanceolata L.) on plasma glucose metabolism in sheep taken from a thermoneutral environment and exposed to a hot environment. The sheep were fed either mixed hay (MH) of orchardgrass (Dactylis glomerata L.) and reed canarygrass (Phalaris arundinacea L.) at a 60:40 ratio or MH and plantain (PL) at a 9:1 ratio in a crossover design for each 23-day period. In both dietary treatments the metabolizable energy (ME) and crude protein intake were designed to be isoenergetic and isoproteinous at around maintenance level. The sheep were taken from a thermoneutral environment (20°C, 70% RH) and exposed to a hot environment (28
30°C, 70% RH) for 5 days. The isotope dilution method using a single injection of [6, 6-2H]glucose was performed on the 18th day of the thermoneutral environment and on the 5th day of heat exposure. Plasma glucose pool size was numerically lower (p = 0.26) during heat exposure on both dietary treatments, and numerically higher (p = 0.13) on the MH diet irrespective of environmental temperature.
Plasma NEFA concentration (p = 0.01) and glucose turnover rate (p = 0.03) were decreased during heat exposure, but remained similar between diets. It could be concluded that, although no positive impact of plantain on glucose metabolism was found under the present experimental conditions (plantain constituted only 10% of basal diet), plantain herb is an alternative to MH for rearing sheep in both thermoneutral and hot environments. (Key Words : Plantain Herb, Heat Exposure, Stable Isotope, Glucose Metabolism, Sheep)
INTRODUCTION
Heat exposure is amajor constraint which reduces the productivity of ruminants through reduced feed intake, elevatedbody temperature, impaired prenatal function and prenatal development. Heat exposure also influences the concentrations of blood metabolites and modifies endocrine secretions (Bell et al., 1987;Bell et al., 1989; Achmadi et al., 1993; Itoh et al., 2001).
The use ofantibiotic growth promoters in animal feed riskspossible drug resistance inhuman pathogenic bacteria.
As a result, feed manufacturers and animal producers are concentratingon natural herbs as analternative to synthetic antibiotics. Plantain (Plantago lanceolata L.) is one of the perennial herbs havingsome bioactive compounds such as acteoside, aucubin, and catalpol (Ishiguro et al., 1982;
Nishibe and Murai, 1995), which have anti-oxidative activity (Zhou and Zheng, 1991; Wang et al., 1996) and
anti-inflammatoryeffects (Muraiet al., 1995; Marchesanet al., 1998).
In ruminants dietary carbohydrates are fermented to volatile fatty acids, the major energy source, by microorganisms in the rumen. Therefore, little glucose is absorbed from the digestive tract and must be supplied through gluconeogenesis. Glucose metabolism isinfluenced by nutritional and physiological conditions (Evans and Buchanan-Smith, 1975; Buckley et al., 1982; Sano et al.,
1983). Sano et al. (1983) also reported that glucose metabolism wasreducedduring heat exposure.
Therefore, it was expected that, due to the presence of bioactive components, plantain might influence the responses to glucose metabolism through reducing heat stress inruminants. However, until now, to the bestof our knowledge,the effect of plantain herbduring heat exposure on intermediary metabolism of glucose in sheep has not been reported.
The present experimentwas conductedtodetermine the effect ofplantain on plasma glucose metabolism in sheep during heat exposure (30°C and 70% relative humidity (RH)), using an isotope dilution method with [6, 6- 2H]glucose.
MATERIALS AND METHODS
Anim지s, diets,andheatexposure
Sixcrossbred (Corriedalex Suffolk) shorn sheep of both sexes (Ovis aries L.), aged around 2 years andwith mean body weight(BW) of 30±2 kg were used. The sheep were housed in individual pens in a barn during thefirst2-week adjustment period of the experiment. Two dietary treatments were used: (1) mixed hay (MH diet) of orchardgrass (Dactylis glomerata L.) and reed canarygrass (Phalaris arundinacea L.) (60:40, 1.78 kcal ME/gair dry matter (DM), 9.8% crude protein (CP)); (2) MH: plantain (PL diet, 9:1). The metabolizable energy (ME) and crude protein intakes were designed to maintain around maintenance level (NRC, 1985) onbothdietarytreatments.
Theanimalswere fed once dailyat 14:00h, and usually ate everything within 1 h,although the manger was untouched tillnextmorning. Water wasavailable ad libitum.
Theexperimentwas performed using a crossover design with two 23-day periods in which either MH or PL diets were fed. Threesheepwere fed the MH diet duringthefirst periodand thenthe PL diet duringthe second period, and the otherthree sheep were fedin thereverse order. Onday 14, the animals were moved to metabolism cages in a controlled environment chamber at an air temperature of 20±1°C, 70% relative humidity (RH) and with lighting from 08:00hto22:00h. Anisotope dilution method using[6, 6- 2H]glucose wasconductedonday 18 ofeach dietary period to determine plasma glucose metabolism in the thermoneutral environment (20±1°C). The environmental temperature was then elevated and maintained at 28-30°C, 70% RH with lighting from 08:00 h to 22:00 h for 5 days, and thesameisotope dilution methodwas performed onthe 5th day of heat exposure. The sheep were weighed at the start of the experiment, on day 14 of the experiment and after finishingeach environmentaltreatment.
Two catheters, one for isotope infusionand another for blood sampling, wereinsertedinto the leftand rightjugular veins on the morning of each isotope dilution procedure.
Catheters were filled with sterile solution of trisodium citrate (0.13 mol/L). Blood sampling was performed without noticeable stress to the sheep. The handling of animals, including cannulation and blood sampling, was carried out according to the rules and regulations established by the Animal Care Committee of the Iwate University.
Experimentalprocedures
Rectaltemperaturewas measuredat 14:00 h for 3 days during both thermoneutral and heat exposure. At 12:00 h on the day of the isotope dilution method, 100 卩mol of [6, 6- 2H]glucose (D-glucose, 6, 6-D2, 99atom%excessD, Isotec,
A Matheson, USACo., Miamisburg, USA) dissolved in 10 ml of saline solution (0.15 mol sodium chloride/L) was injected through the jugular infusion catheter. Blood samples (5 ml) were collectedfrom the sampling catheter immediatelybefore and at 20, 40,60, 90 and 120minafter the isotope injection for the determination of plasma glucose concentration, pool size and turnover rate. Samples were transferred intoheparinized centrifuge tubes and were chilleduntil centrifugation.
An이yses
Bloodsampleswere centrifugedat 12,000xg for10 min at 2°C (RS-18 IV, Tomy, Tokyo, Japan), and the plasma was stored at -30°C until further analysis. Isolation and derivatization of plasma glucose was performed by the procedure of Tserng andKalhan (1983) and Wolfe (1984) with slight modificationby Fujita et al.(2007). The isotopic abundance of the glucose derivative was determinedwith a gas chromatography mass spectrometry system (QP-2010, Shimadzu, Kyoto, Japan) with selected ion monitoring.
Concentration of plasma glucose was enzymatically determined by the method of Huggett and Nixon (1957).
Concentration ofplasma nonesterified fatty acids (NEFA) was enzymatically determined as described previously (Fujita et al., 2006) with akit (NEFA C test, Wako Pure Chemicals,Osaka, Japan).
Calculations
Plasma glucose pool size and turnover rate were calculatedaccording toa single-poolanalysis (Wolfe, 1984).
The sampled pool represents the blood, which is in rapid equilibrium with the interstitial fluid (Schmidt and Keith,
1983).
Statisticalanalysis
All data were analyzed withthe MIXED procedure of SAS (1996). The lsmeans statement was used to test the effects of period, diet, environment andinteraction of diet and environment. The random effect was sheep. Results were considered significant at the p<0.05 level, and a tendency was defined as 0.05<p<0.10. The repeated statement and the Tukey-Kramer adjustment were used for the time course of changes and the significance level was p<0.05 and p<0.10,respectively.
RESULTS
Dailyprofiles
Body weight of animals on MH and PL diets remained unchanged in both environments. Rectal temperature was higher (p = 0.007) during heat exposure on bothdiets and there was a trend to higher (p = 0.09) on MH dietthan PL
Table 1. Eiiects of plantain supplementation and heat exposure on rectal temperature and plasma non-esterified fatty acid (NEFA) concentration in sheep
* MH =Orchardgrass(Dactylis glomerata L.) and reed canarygrass (Phalaris arundinacea L.) hay (60:40).
PL =Orchardgrass (Dactylisglomerata L.) andreedcanarygrass (Phalaris arundinacea L.)hay mixture and plantain(9:1 ratio).
TN = Thermoneutral (20°C); Heat= Heat exposure (28-30°C); Env = Environment; DietxEnv = Diet and environment interaction.
SEM =Standarderror of the mean.
MH* PL
SEM Significance
TN Heat TN Heat Diet Env DietxEnv
No. of sheep 6 6 6 6
Rectal temperature (°C) 39.6 40.0 39.4 40.0 0.1 0.53 0.007 0.09
NEFA (mmol/L) 0.24 0.18 0.25 0.15 0.03 0.75 0.01 0.45
Table 2. Effects of plantain supplementation and heat exposure on concentration, pool size and turnover rate of plasma glucose in sheep
MH* PL
SEM Significance
TN Heat TN Heat Diet Env DietxEnv
No. of sheep 6 6 6 6
Glucose concentration (mmol/L)** 3.4 3.4 3.5 3.2 0.03 0.47 0.13 0.20
Pool size (mmol/kg BW0.75) 2.3 2.1 2.1 1.9 0.1 0.13 0.26 0.95
Turnover rate (mmol/kg BW0.75/day) 37 32 40 33 2 0.52 0.03 0.92
* MH =Orchardgrass (Dactylisglomerata L.)and reed canarygrass (Phalaris arundinacea L.) hay (60:40).
PL =Orchardgrass(Dactylis glomerata L.) and reed canarygrass (Phalaris arundinacea L.) hay mixture and plantain(9:1 ratio).
TN = Thermoneutral(20°C);Heat = Heatexposure (28-30°C); Env = Environment;DietxEnv= Diet and environment interaction.
SEM = Standard error of the mean.
** Mean values ofthe plasmaglucose concentrations during 120 min of each experiment.
븡(
lulu)
oc oo
(%)-u을
흐
Juo
% 3 - 으u -s d
g 3 -
"
H 9
。으 4.5 [
3.冲~
7 -
A I2.5 L
Figure 1. Time course of changes in plasma glucose concentrations and plasma [6, 6-2H]glucose enrichments after a single injection of [6, 6-2H]glucose in sheep fed the MH diet in thermoneutral and heat exposure (open triangle and square, respectively) and PL diet in thermoneutral and heat exposure (solid triangle and square, respectively).
diet (Table 1). Plasma NEFA concentration decreased during heat exposure (p = 0.01), and remained similar between diets.
Blood glucose metabolism
Plasma glucose concentrations remained constant
during the 120 min period in each experiment (Figure 1).
Plasma [6, 6-2H]glucose enrichmentsdecreased as asingle
exponential function from20 min to 120 minafter [6, 6- 2H]glucose injection. Plasma glucose concentrations were unchangedwith dietary and environmentaltreatments (p= 0.47 and p = 0.13, respectively, Table 2). Plasma glucose pool size was numerically lower (p = 0.26) during heat exposure inboth dietary treatments, and numerically higher (p = 0.13) on MH diet irrespective of environmental temperature. Plasma glucose turnover rate decreased (p = 0.03) during heat exposure, but remained similarbetween diets (p= 0.52).
DISCUSSION
In the present study rectal temperature increased (p = 0.007) during heat exposure which agrees with previous findings (Achmadi et al., 1993; Itoh et al., 2001). Plasma NEFA concentrations decreased duringheat exposure (p = 0.01), but aneffect ofplantain was not observed (p = 0.74).
However, plasma NEFA concentrationtended tobe lower in sheep fed the plantain dietthan for the orchardgrass diet (Sano etal., 2002). The inconsistency might be due to the small amount of plantain (10% of MH was replaced by plantain) used inthe present experiment. In another study, Sano et al. (1983) reported that plasma NFFA concentrations decreased during heat exposure in sheep, which agrees with our findings. They also reported that concentrationof plasmathyroxine decreased and respiration rate increased markedly during heat exposure. Therefore, considering the physiological responses and blood
constituents, sheep were assumed to be influenced by heat exposure.
The plasma glucose concentrations were unchanged (p
=0.13) during heat exposure and between diets (p = 0.47) inthe present experiment. Basalglucose concentration was not significantly lower duringheat (28°C) exposure (Itohet al., 1998) in lactating dairy cows administered i.v. with glucose, arginine and butyrate, which agrees with the present findings. However, plasma glucose concentrations were lower (p<0.01) in sheep during heat exposure as mentioned previously (Achmadi et al., 1993). The contrast may partly be due to a difference of dietary compositionas these authors fed a diet containing a large proportion of concentrate. It has been found that lower forage to concentrate ratios in diets increasesthe efficiency of energy utilization during heat exposure (Beede and Collier, 1986).
It has also been reported that basal glucose concentration decreases duringheat exposure (30°C) in ewes fed alfalfa hay cubes at maintenance level (Itoh et al., 2001). The inconsistency of these findings might be related to animal species, physiological and environmental conditions, feed composition and feeding regimen.
A number of experiments to determine glucose metabolismhavebeendoneusing either thesingle injection or primed continuous infusion methods of glucose isotope dilution (Kronfeldand Simesen, 1961; Buckley etal., 1982;
Sano et al., 2007). In the present study we usedthe single injection method of [6, 6-2H]glucose dilution, because, unlike primed continuous infusion, it allows measurement ofplasma glucose pool size in addition to glucose turnover rate. However, Buckley et al. (1982) applied both single injection of [6-3H]glucose and the primed-continuous infusion of [U-13C]glucose for determination of glucose metabolism in lactating and non-lactating goats, and reported that glucose irreversibleloss rates were about 20%
greater for the single injectionmethod. However, the values of thisparameterdetermined in the present experimentwere comparable between treatments. In the present study, the numerical values ofplasma glucose pool size were lower during heat exposure in both dietary treatments. In a previous study,plasma glucose pool sizetendedtodecrease during heat (30°C) exposure in sheep (Sano et al., 1979) which agrees with the present findings. The meanvalues of theplasma glucose pool size in sheep, fedalfalfa hay cube and concentrate mixture twice daily, in a previous study (Sano et al., 1979) were slightly higher than the values found in the present experiment. This might be due to differences in the intake,feeding regime and frequencyand composition of feed. However, plasma glucose pool size remainedunchanged after feedrestriction inlactating ewes (Gow et al., 1981), and glucose pool size was hardly influenced by energy and dry matter intake (Schmidt and
Keith, 1983).
Blood glucose turnover rate decreased during heat exposure (p = 0.03) on both diets, although dietary intake was notinfluenced. Similar results were observed in sheep fed alfalfa hay cube and commercial concentrate (1:1) during heat exposure (30°C) (Sano et al., 1983), and in sheepexposedto 30°C and fed lucerne hay with chromium supplementation (Sano et al., 2000). Whole-body glucose kinetics are alsoinfluencedpartiallybyendocrinehormones.
Synthesis ofthyroxineand triiodothyroninewerereduced in lactating cows when exposed to heat (31.2°C)for 10 days (Magdub et al., 1982), and concentrationof thyroxine in plasmawas decreased in sheepduring heat exposure (Sano et al., 1983). Triiodothyronine concentration is directly related to glucose utilization (Saunders et al., 1978), which is regulated by the activities of glycolytic enzymes (Lombardi et al., 2000). Whole-body glucose utilization in sheep and glucose utilization in adiposetissue of fat-fedrats werestimulated by epinephrine (Susinietal., 1979; Sano et al., 1996). Epinephrine secretionis expected to have been reduced during heat exposure, because plasma NEFA, which ismobilized by epinephrine,wasreducedduringheat exposure. Blood glucose turnover ratemaybe influenced by hormones as well as type of diet, energy intake, and physiological status (Evans and Buchanan-Smith, 1975;
Weekes, 1979; Janes et al., 1985; Ortigues-Marty et al., 2003; Sano et al., 2007).
Fewexperimentshavebeenconductedto studyglucose metabolism in animals fed plantain herb, except for an experiment by Sano et al. (2002) in which glucose metabolism in sheep was studied using [U-13C]glucose.
Although not significant, the numerical values of glucose turnover rate were higher for the PL diet in both the thermoneutral and hot environments in the present experiment. This mightpartly be relatedto the availability of volatile fatty acids as the energy source. The concentrationof propionate, a major glucose precursor, in the rumen was higher on the plantain diet than on the orchardgrass diet in sheep whereas acetate concentration decreased slightly (Sanoet al., 2002).
It couldbe concluded that plasma glucose metabolism was influenced by heat exposure and remained similar between diets. No significant interaction between diet and environments was observed in relation to glucose turnover rate,suggesting that plantainherb iscomparableto MH diet in both thermoneutral and hot environments. It was hypothesized that the bioactive components present in plantain could improve the metabolism but no positive impactwas found underthe present experimentalconditions (only 10% of MHwas replacedby plantain). Thus, further investigations need to be performed with an increasing amount of plantain in the basaldiet.
REFERENCES
Achmadi, J., T. Yanagisawa, H. Sano and Y. Terashima. 1993.
Pancreatic insulin secretory response and insulin action in heat-exposed sheep given a concentrate or roughage diet.
Domest. Anim. Endocrinol. 10:279-287.
Beede, D. K. and R. J. Collier. 1986. Potential nutritional strategies for intensively managed cattle during thermal stress.
J. Dairy Sci. 62:543-554.
Bell, A. W., B. W. McBride, R. Slepetis, R. J. Early and W. B.
Currie. 1989. Chronic heat stress and prenatal development in sheep: I. Conceptas growth and maternal plasma hormones and metabolites. J. Anim. Sci. 67:3289-99.
Bell, A. W., R. B. Wilkening and G. Meschia. 1987. Some aspects of placental function in chronically heat stressed ewes. J. Dev.
Physiol. 9:17-19.
Buckley, B. A., J. H. Herbein and J. W. Young. 1982. Glucose kinetics in lactating and nonlactating dairy goats. J. Dairy Sci.
65:371-384.
Evans, E. and J. G. Buchanan-Smith. 1975. Effects upon glucose metabolism of feeding a low-or high roughage diet at two levels of intake to sheep. Br. J. Nutr. 33:33-44.
Fujita, T., T. Itoh, H. Majima and H. Sano. 2007. Influence of dietary salinomycin on feeding-induced variations of glucose kinetics and blood volatile fatty acids and insulin concentrations in sheep fed a high-roughage diet. Asian-Aust. J.
Anim. Sci. 20:365-372.
Fujita, T., M. Kajita, H. Sano and A. Shiga. 2006. Effects of non
protein energy intake on the concentrations of plasma metabolites and insulin, and tissue responsiveness and sensitivity to insulin in goats. Asian-Aust. J. Anim. Sci.
19:1010-1018.
Gow, C. B., G. H. McDowell and E. F. Annison. 1981. Control of gluconeogenesis in the lactating sheep. Aust. J. Biol. Sci.
34:469-478.
Huggett, A. G. and D. A. Nixon. 1957. Enzymatic determination of blood glucose. Biochem. J. 66:12.
Ishiguro, K., M. Yamaki and S. Takagi. 1982. Studies on the iridoid related compounds. I.On the antimicrobial activity of aucubigenin and certain iridoid aglycones. Yaku. Zas. 102:755
759.
Itoh, F., K. Hodate, S. Koyama, M. T. Rose, M. Matsumoto, A.
Ozawa and Y. Obara. 2001. Effects of heat exposure on adrenergic modulation of insulin and glucagons secretion in sheep. Endocrine J. 48:193-198.
Itoh, F., Y. Obara, M. T. Rose, H. Fuse and H. Hashimoto. 1998.
Insulin and glucagon secretion in lactating cows during heat exposure. J. Anim. Sci. 76:2182-2189.
Janes, A. N., T. E. C. Weekes and D. G. Armstrong. 1985.
Absorption and metabolism of glucose by the mesenteric- drained viscera of sheep fed on dried-grass or ground, maize
based diets. Br. J. Nutr. 54:449-458.
Kronfeld, D. S. and M. G. Simesen. 1961. Glucose biokinetics in sheep. Am. J. Physiol. 201:639-644.
Lombardi, A., L. Beneduce, M. Moreno, S. Diano, V. Colantuoni, M. V. Ursini, A. Lanni and F. Goglia. 2000. 3,5-Diiodo-L- thyronine regulates glucose-6-phosphate dehydrogenase activity in the rat. Endocrinol. 141:1729-1734.
Magdub, A., H. D. Johnson and R. L. Belyea. 1982. Effect of environmental heat and dietary fiber on thyroid physiology of lactating cows. J. Dairy Sci. 65:2323-2331.
Marchesan, M., D. H. Paper, S. Hose and G. Franz. 1998.
Investigation of the anti-inflammatory activity of liquid extracts of Plantago lanceolata L. Phytoth. Res. 12:S33-S34.
Murai, M., Y. Tamayama and S. Nishibe. 1995. Phenylethanoids in the herb of Plantago lanceolata and inhibitory effect on arachidonic acid-induced mouse ear edema. Planta Med.
61:479-480.
Nishibe, S. and M. Murai. 1995. Bioactive components of Plantago herb. Foods Food Ingr. J. 166:43-49.
National Research Council. 1985. Nutrient requirement of sheep.
6th edn. National Academy Press. Washington, DC.
Ortigues-Marty, I., J. Vernet and L. Majdoub. 2003. Whole body glucose turnover in growing and non-productive adult ruminants: meta-analysis and review. Reprod. Nutr. Dev.
43:371-383.
Sano, H., T. Fujita, M. Murakami and A. Shiga. 1996. Stimulative effect of epinephrine on glucose production and utilization rates in sheep using a stable isotope. Domest. Anim.
Endocrinol. 13:445-451.
Sano, H., K. Takahashi, M. Fujita, K. Ambo and T. Tsuda. 1979.
Effect of environmental heat exposure on physiological responses, blood constituents and parameters of blood glucose metabolism in sheep. Tohoku J. Agric. Res. 30:76-86.
Sano, H., S. Konno and A. Shiga. 2000. Effects of supplemental chromium and heat exposure on glucose metabolism and insulin action in sheep. J. Agric. Sci. 134:319-325.
Sano, H., K. Takahashi, K. Ambo and T. Tsuda. 1983. Turnover and oxidation rates of blood glucose and heat production in sheep exposed to heat. J. Dairy Sci. 66:856-861.
Sano, H., H. Sawada, A. Takenami, S. Oda and M. Al-Mamun.
2007. Effects of dietary energy intake and cold exposure on kinetics of plasma glucose metabolism in sheep. J. Anim.
Physiol. Anim. Nutr. 91:1-5.
Sano, H., Y. Tamura and A. Shiga. 2002. Metabolism and glucose kinetics in sheep fed plantain and orchardgrass and exposed to heat. N.Z. J. Agric. Res. 45:171-177.
SAS Institute Inc. 1996. SAS/ STATR Software: Changes and Enhancements through Release 6.11. SAS Institute Inc., Cary, NC.
Saunders, J., S. E. H. Hall and P. H. Sonksen. 1978. Thyroid hormones in insulin requiring diabetes before and after treatment. Diabetologia 15:29-32.
Schmidt, S. P. and R. K. Keith. 1983. Effects of diet and energy intake on kinetics of glucose metabolism in steers. J. Nutr.
113:2155-2163.
Susini, C., M. Lavau and J. Herzog. 1979. Adrenaline responsiveness of glucose metabolism in insulin-resistant adipose tissue of rats fed a high-fat diet. Biochem. J. 180:431
433.
Tserng, K.-Y. and S. C. Kalhan. 1983. Calculation of substrate turnover rate in stable isotope tracer studies. Am. J. Physiol.
245:E308-E311.
Wang, P., J. Kang, R. Zheng, Z. Yang, J. Lu., J. Gao and Z. Jia.
1996. Scavenging effects of phenylpropanoid glycosides from pedicularis on superoxide anion and hydroxyl radical by the
spin trapping method (95) 02255-4. Biochem. Pharmacol.
51:687-691.
Weekes, T. E. C. 1979. Carbohydrate metabolism. In: Digestive physiology and nutrition of ruminants. (Ed. D. C. Church).
Corvallis O & B Books Inc. pp. 187-209.
Wolfe, R. R. 1984. Tracers in Metabolic Research. Alan R. Liss, Inc., New York.
Zhou, Y. C. and R. L. Zheng. 1991. Phenolic compounds and an analog as superoxide anion scavengers and antioxidants.
Biochem. Pharmacol. 42:1177-1179.
